A SINGLE AMINO ACID SUBSTITUTION (ASPARAGINE TO ASPARTIC ACID) BETWEEN NORMAL (B+) AND THE COMMON NEGRO VARIANT (A+) OF HUMAN GLUCOSE-6-PHOSPHATE DEHYDROGENASE* BY AKIRA YOSHIDA DIVISION OF MEDICAL GENETICS, DEPARTMENT OF MEDICINE, UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE, SEATTLE Communicated by C. B. Anfinsen, January 24, 1967 Extensive work with human hemoglobins indicates that the typical variant hemo- globin differs from normal hemoglobin by a single amino acid substitution. MAuta- tional studies with E. coli tryptophan synthetase have shown that variants of this protein also owe their origin to single replacement of an amino acid. Using bio- chemical and genetic recombination techniques, it has been shown that the site of such replacements in the amino acid of the enzyme bears a linear relationship to the site of the mutation within the gene specifying the structure of this enzyme.I Based on this type of evidence, there is general belief among biochemical geneticists that most mutationally altered enzymes are produced by single amino acid sub- stitutions. Since it is difficult to obtain the necessary quantities of pure enzyme proteins for the required biochemical analyses, proof for this hypothesis for mutant enzymes from mammals is not yet available. About 20 variants of human red cell glucose-6-phosphate dehydrogenase (D- glucose-6-phosphate: NADP oxidoreductase, E.C.1.1.1.49) are known. A com- mon variant found in about 18 per cent of American Negro males manifests with rapid electrophoretic mobility and is not associated with enzyme deficiency.2-4 Normal human glucose-6-phosphate dehydrogenase (B+) and the Negro-type variant with normal activity (A+) have beet isolated in homogeneous form, and their molecular weights, subunit molecular sizes, amino acid compositions, enzy- matic properties, and serological characteristics have been compared.5' 6 The results indicated that the two enzymes were very similar and that any structural difference between them would be very small. Further studies indicate that the two enzymes differ in a single amino acid-asparagine to aspartic acid. AMaterials and Methods. -Glucose-6-phosphate dehydrogenase: The normal (B+) and the Negro-type variant (A+) enzymes were prepared from blood by methods described previously.5 6 The preparations were homogeneous by physicochemical criteria. Trypsin (EC.3.4.4.4, salt-free, 3 X cryst.), leucine amino peptidase (EC.3.4. 1.1, diisopropylfluorophosphate, DJF1P treated, 80 units/mg), carboxy- peptidase A (EC.3.4.2.1, 2X cryst., 40 units/mg), and carboxypeptidase B (EC.- 3.4.2.2, 100 units/mg) were purchased from Worthingtonl Biochemical Corporation. Leucine amino peptidase was activated by i\4gCl2 before use. Carboxypeptidase A and B were treated with DFP before use.7 Urea was deionized by recrystallization according to the method described by P enesch et al.8 lodoacetic acid was recrystallized from ether and kept at -200. The procedures used for reduction and S-carboxymethylation of the enzyme were essentially those described by Crestfield et al.,9 and the details have been reported. 1 Trypsin digestion: The lyophilized powder of the reduced S-carboxymethylated protein was digested by trypsin at room temperature for four hours. During the 835 Downloaded by guest on September 27, 2021 836 BIOCHEMISTRY: A. YOSHIDA PROC. N. A. S. digestion, the pH of the reaction mixture was maintained at pH 8.0-8.2 by adding 0.01 M (NH4)HCO3. The reaction mixture was acidified to pH 3-4 with acetic acid and lyophilized. Fingerprinting: Tryptic hydrolysates (lyophilized powder), 2.5-3 mg, were applied to a Whatman no. 3 MM (57 X 46 cm paper). The method of fingerprint- ing of the tryptic hydrolysates was essentially that described by Katz et al.I Pep- tide spots were developed by heating at 600 for 30 minutes after spraying 0.025 per cent of ninhydrine solution in acetone. Results. -Fingerprints: The fingerprints of tryptic peptides obtained from the normal (B+) and the Negro-type variant (A+) of glucose-6-phosphate dehydro- genase are shown in Figure 1. Among approximately 40 peptide spots appearing in each fingerprint, only one peptide spot (marked I) of the normal enzyme was re- placed by a peptide (marked II) in the variant enzyme; all other peptide spots cor- responded to each other in both enzymes. The results strongly suggested a single amino acid substitution between the normal and the variant enzyme. Since peptide I moved more rapidly than peptide II on electrophoresis at pH 3.6-3.7, it was likely that an amino acid residue of peptide I might be substituted by a more acidic amino residue of peptide II. Amino acid substitution: The peptide spots marked I and II were cut and eluted. Amino acid analysis was carried out with a Technicon amino acid analyzer. The amino acids found in peptides I and II are shown in Table 1. Both peptides had the same amio acid composition after acid hydrolysis. Considering the difference of electrophoretic mobility, asparagine or glutamine in peptide I should be replaced by aspartic acid or glutamic acid in peptide II, since asparagine and glutamine are converted to aspartic acid and glutamic acid by acid hydrolysis. To distinguish between the two possibilities, peptides I and II were digested by peptidases, and the amino acids that appeared were identified by high-voltage electrophoresis and paper chromatography. Each peptide (I or II) was dissolved in 0.25 ml of 0.1 M triethanolamine- acetic acid buffer (pH 7.9) containing 0.005 M MgCl2 and was digested by leucine amino peptidase (0.021 mg) at 30° for 20 hours (toluol was added to prevent bacterial growth). Subsequently, the peptide was digested with carboxypeptidase B (0.05 mg) and carboxypeptidase A (0.01 mg) for another 20 hours at 300. As a control, the reaction mixture without the peptide was incubated under the same conditions. After digestion, the reaction mixture was acidified to pH 3-4 with acetic acid and treated with about 0.3 ml (bed volume) of Dowex-50, X 12 (H+ form, 20-50 mesh). The amino acids absorbed by the resin were extracted with 4 N NH40H. The eluents were evaporated to dryness under reduced pressure in the presence of sul- furic acid and used for identification of amino acids. The analysis was carried out using the following system: first dimension: paper electrophoresis, Whatman no. 3MM (57 X 46 cm), formic acid-pyridine- water (4:03:96, V/V, pH 2.2), 2000 volts for 60 miin; second dimension: paper chromatography, ethanol-water (77:23, V/V) for 16 hours. This system gave a good separation of all amino acids which might be contained in the peptides I and II. Thus, it was possible to distinguish asparagine, aspartic acid, glutamine, and glutamic acid. Downloaded by guest on September 27, 2021 VOL. 57, 1967 BIOCHEMISTRY: A. YOSHIDA 937 FIG. 1.-Fingerprint of tryptic digest of reduced, S-carboxymethylated human glucose-6-phos- phate dehydrogenase. I, Normal enzyme; II, Negro-type variant. First dimension, chroma- tography, n-butanol-acetic acid-water (4:1:5, upper layer), for 18Ihr. 'Second dimension, elec- trophoresis, pyridine-acetic acid-water (1: 10:289, pH 3.6-3.7), 2000 volts for 60 min. Downloaded by guest on September 27, 2021 8,388BIOCHEMISTRY: A. YOSHIDA PROC. N. A. S. The hydrolysates of peptide I contained very little aspartic acid (apparently in the same order of the control) and a large quantity of asparagine. Peptide II con- tained both asparagine and aspartic acid in apparently the same quantity. Both peptide hydrolysates contained glutamine instead of glutamic acid. From these findings, together with the results of amino acid composition of acid hydrolysates of the peptides (Table 1), the following amino acid compositions for the peptides I and II were established: Peptide I (from the normal enzyme): Asn2, Seri, Glni, Glyl, Ala2, Leu2, His2, Argi. Peptide II (from the Negro-type variant): Asni, Asp,, Seri, Gln1, Gly1, Ala2, Leu2, His2, Argi. Accordingly, the amino acid substitution between the two enzymes is asparagine (normal enzyme) to aspartic acid (Negro-type variant). Discussion.-About 40 peptide spots appeared (Fig. 1) in the fingerprints of the normal or the Negro-type variant of human glucose-6-phosphate dehydrogenase. From the number of peptide spots and the known molecular weight, as well as the content of lysine and arginine in the protein, one can estimate the number of sub- units if the following reasonable assumptions are met: (1) nearly all the trypsin- susceptible peptide bonds of the denatured, reduced and S-carboxymethylated protein are hydrolyzed under the conditions used; (2) digestion by other proteolytic enzymes which may contaminate the trypsin or by chymotrypsin-like activity of trypsin itself is negligible; (3) the resolution of the fingerprint is high enough to separate nearly all tryptic peptides. The molecular weight of human glucose-6-phosphate dehydrogenase is about 230,000-240,000, and the content of lysine residue is 6.20 + 0.01 per cent (110-115 residues per molecule) and that of arginine residues is 8.04 1 0.30 per cent (125- 130 residues per molecule).5 6 From the number of peptide spots observed on the fingerprint, it can be concluded that the protein consists of six repeating units (mol wt = 40,000) since the expected number of tryptic peptides in a protein of molecular weight of 40,000 is about 40 (about 19 lysine residues and about 22 arginine residues per subunit). It has been found that the enzyme dissociated into smaller subunits with a mo- lecular weight of about 43,000 in the presence of 4 M guanidine-HCl and mercapto- ethanol.5' 6 Therefore, the molecular size of the repeating unit found by finger- printing is consistent with the molecular size of these subunits found by the sedi- TABLE 1 AMINO ACID COMPOSITION OF TRYPTIC PEPTIDE OF HUMAN GLUCOSE-6-PHOSPHATE DEHYDROGENASE Normal enzyme, Negro-type variant, peptide I peptide II Ratio of amino Amino acid (Mmole)* (umole)* acid residuest Aspartic acid 0.043 0.040 2 Serine 0.021 0.020 1 Glutamic acid 0.019 0.016 1 (Glycine 0.020 0.018 1 Alanine 0.032 0.032 2 Leucine 0.039 0.038 2 Histidine 0.036 0.033 2 Arginine 0.021 0.018 1 * Amount of amino acid found in peptide I and peptide II eluted from one fingerprint each.